Diabetes is a serious and prevalent disease. In the United States alone, over two million people have Type I, Insulin Dependent Diabetes Mellitus, and about eight to ten million people have Type II, non-Insulin Dependent Diabetes Mellitus. Microvascular complications caused by the disease make it the third leading cause of death, the leading cause of new blindness, the cause of kidney failures resulting in 30% of new kidney transplants, a major factor in myocardial infarction and stroke, a leading cause of gangrene and amputation, and an important cause of male impotence. The total monetary impact of the disease in the United States alone is estimated to be around $14 billion a year.
Insulin is produced by islets of Langerhans located in the pancreas. The isolation of islets from a donor pancreas or from an animal pancreas, and the transplantation of those islets into a diabetic patient, may be a viable treatment for diabetes.
By transplanting only purified islets rather than intact pancreatic segments, the transplantation can be accomplished by injecting the islets into the bloodstream so that they lodge in the liver rather than by surgically implanting the pancreatic segments into the patient's pancreas. Intravenous injection of the islets also allows that possibility of treating the islets in vitro to prevent rejection without the necessity of immunosuppression, it avoids problems associated with the secretion of digestive substances by a transplanted pancreas segment, and it allows the possibility of cryopreservation of the tissue for later use and the possibility of xenografts.
The removal of islets from the pancreas to obtain a tissue suspension of islets and pancreatic tissue has been explored at length. The typical procedure is to break down the pancreatic tissue with a digestive enzyme such as collagenase to free the islets. Such procedures are described in a number of scientific publications, including World Journal of Surgery, April 1984 and the Journal of the American Diabetes Association, Vol. lb, No. 1, pp 35-39 "Method for the Isolation of Intact Islets of Langerhans from the Rat Pancreas."
The difficulty with collagenase digestion of the pancreatic tissue to free the islets is that the individual islets are freed at different rates based on their size distribution, concentration and degree of entrainment in the tissue. Therefore, during the time the collagenase is digesting pancreatic tissue to free the unfreed islets, it is also continuing to act on the islets that have already been freed, thereby breaking up the freed islets into smaller groups and even individual cells and degrading those cells. The end result is that the number of viable islets that are freed by this process is much less than the number of islets in the pancreatic sample that is processed.
There has been considerable effort directed toward modifying the basic collagenase digestion process in an attempt to achieve higher effective separation rates. These efforts have included, for example, the use of VELCRO brand hook fasteners to collect tissue fragments (see Diabetes, Vol. 31, Suppl. 4, August 1982, "An Improved Method for the Isolation of Islets from the Beef Pancreas"); teasing and shaking the pancreas to assist in releasing the islets (see Diabetes, Vol. 33, November 1984, "A Method for Isolation of Islets of Langerhans from the Human Pancreas"); dicing the pancreas into small units (see Transplantation Proceedings, Vol. XVII, No. 1 (February), 1985, "Isolation of Human Pancreatic Islets from Cryopreserved Pancreas"); methods for minimizing gelation in the pancreas which tends to trap islets (see Diabetes, Vol. 38, Suppl. 1, January 1989, "Factors Influencing Isolation of Islets of Langerhans"; and controlling the collagenase contents (see Diabetes, Vol. 38, Suppl. 1, January 1989, "Protease Activity in Pancreatic Islet Isolation by Enzymatic Digestion").
Another of these methods to increase islet yield relating to collagenase digestion relies on injecting the collagenase into the pancreas main duct. Such a method using continuous peristaltic ductal perfusion is taught in Transplantation Proceedings, Vol. 22, No. 2 (April), pp. 789-790, "A Simple Method for Bulk Separation of Highly Purified Human Islets of Langerhans". A similar method using spherical agitators in a container to enhance tissue agitation is described in U.S. Pat. No. 4,868,121 by Scharp et al., and in PCT International Application No. WO 88/09667, published Dec. 15, 1988, naming Washington University as Applicant.
Once the islets are separated from the pancreas and isolated in a solution containing islets and partially digested pancreas tissue fragments, there are several techniques for concentrating and purifying the tissue suspension. The most common includes concentrating the solution and then centrifuging the solution with a medium solution such as a Ficoll or Percoll gradient so that the islets can be isolated and drawn off from the solution and from the tissue fragments. Such methods are described in some detail in, for example, Transplantation, August 1976, pp. 201-205, "The Use of Hypaque-Ficoll in the Isolation of Pancreatic Islets in Rats"; Biochemical and Biophysical Research Communications, Vol. 79, No. 3, 1977, p. 823, "Rapid Isolation of Pancreatic Islets from Collagenase Digested Pancreas by Sedimentation through Percoll at Unit Gravity"; Endocrinal, 1981, 28(5), pp. 563-567, "A Rapid Method for the Separation of Rat Pancreatic Islets from Collagenase-Digested Pancreas using Percoll"; Transplantation, Vol. 43, No. 6, pp. 805-808, "Bovine Serum Albumin Density Gradient Isolation of Rat Pancreatic Islets"; Transplantation Proceedings, Vol. 22, No. 2 (April), 1990, pp. 758-8759, "Human Pancreatic Islet Isolation with Increased Incubation Temperatures and Variable Density Gradients"; Transplantation Proceedings, Vol. 22, No. 2 (April) 1990, pp. 789-709, "A Simple Method for Bulk Separation of Highly Purified Human Islets of Langerhans."
Other techniques for concentration and purification use filtering, either alone or in combination with centrifuging. See, for example, Diabetes, Vol. 25, No. 8, pp. 667-672, "Standardization of a Digestion-Filtration Method of Isolation of Pancreatic Islets"; Diabetes, Vol. 33, November 1984, "A Method for Isolation of Islets of Langerhans from the Human Pancreas"; Diabetes, Vol. 33, November 1984, "A Method for Isolation of Langerhans from the Human Pancreas"; and Diabetes, Vol. 35, June 1986, "A Method for the Mass Isolation of Islets from the Adult Pig Pancreas". Also, see the Scharp patent and Washington University published PCT application referenced above.
Both the centrifuging and filtering processes have drawbacks. Both are fairly time-consuming. They are also somewhat variable in the results they achieve, in view of the differences normally encountered from batch to batch of islet-containing solutions. Also of importance is that islets are thought to be quite fragile, and both centrifuging and force-filtering may damage the islets and degrade their viability.
As explained in detail below, the present invention partially concentrates the islets by using gravity sedimentation of islets through an inclined channel with a collection well at the bottom. The partially concentrated islets can then be further concentrated with a minimum of ordinary centrifigation or filtering or other processes known in the art. Although the general concept of gravity sedimentation of suspended solids through an inclined channel is well-known, it is believed that such an approach has never been adopted for the processing of islets. U.S. Pat. No. 4,765,899 by Wells et al. describes an apparatus for continuously separating blood components which utilizes a disk-shaped chamber with a central inlet and circumferential upper and lower outlets. In the Wells method, solids do not in fact fall to the bottom of the container, but the fluid is merely stratified into fractions. Moreover, the configuration and flow parameters are designed for blood separation, and nothing in the Wells patent suggests using the Wells apparatus to attempt islet processing. Another patent, U.S. Pat. No. 4,424,132 by Iriguchi, describes a non-inclined chamber having an inlet at one end and upper and lower outlets at the other end to stratify blood components. As in the Wells patent, the configuration and flow parameters are designed for blood separation, and nothing in the patent suggests using any such apparatus for islet processing.